This invention relates to a method of determining spermatozoa qualities, and is particularly useful for determining the viability and fertilising ability of avian spermatozoa.
Methods for assesing the quality of spermatozoa from domestic fowl and other animals, and estimations of their value as predictors of sperm fertilizing ability have been established for many decades (Cooper and Rowell, 1958; Wilson et al., 1979).
Earlier tests (Cooper and Rowell, 1958; Wilson et al., 1979) were perceived to be highly subjective, involving operator-dependent xe2x80x98scoringxe2x80x99 of a particular sperm parameter, with poor agreement between operators. More recently, this criticism has been answered by the introduction and application of a range of objective tests of chicken sperm quality (Bilgilli and Renden, 1985; Wishart and Palmer 1986; Chaudhuri and Wishart, 1988; Froman and McLean, 1996; Barbato et al., 1997). These tests have additionally shown that sperm quality in individual domestic fowl is a xe2x80x98traitxe2x80x99 which is repeatable between ejaculates in the short and longer term and can be linked to fertilizing ability (Wishart and Palmer, 1986; Chaudhuri et al., 1988; Froman et al., 1997; 1998).
A limitation of these objective tests of sperm quality is that they require laboratory facilities such as centrifuges and/or lack the robustness required for use xe2x80x98on the farmxe2x80x99. The INT-tetrazolium dye test introduced by Chaudhuri and Wishart (1988) is relatively robust, and its outcome is easily interpreted as the intensity of colour produced by spermatozoa during a short incubation. INT-reduction was also strongly correlated with other tests of sperm xe2x80x98qualityxe2x80x99, including their fertilizing ability (Chaudhuri et al., 1988). However, reagents involved in the test are unstable and some (e.g. cyanide) are highly toxic. An additional disadvantage is that samples require to be centrifuged before they are sufficiently optically clear to be read with a colorimeter. These problems have limited the practical applicability of the INT-reduction assay.
The present invention provides a method of assessing sperm quality comprising determining whether (and optionally, to what extent) the sperm being assessed can reduce a tetrazolium dye, wherein the dye reduction is determined by monitoring colour changes at a pH above 4.0.
The tetrazolium dye is preferably MTT, which can be added to the sperm in the reaction mixture, or vice versa.
The reaction between the sperm and the dye is preferably carried out for about 1 hr at 37-40xc2x0 C., and preferably at near neutral pH such as pH 6-8, e.g. pH 7.4.
The method preferably includes the step of adding a solubilizing agent such as a detergent such as SDS, or some other agent capable of solubilizing the sperm cells. Solubilizing the sperm cells or otherwise releasing their contents enables colorimetric assessment of the extent of reduction of the tetrazolium dye without centrifugation of the reaction mixture. The solubilizing agent conveniently also stops the reaction.
The colorimetric assessment of dye reduction is preferably carried out as a separate step in slightly more acidic conditions than the reaction of the dye with the sperm, such as pH 5-7 and in an especially preferred embodiment the colorimetric assessment of the dye reduction is carried out at around pH 6.
In a preferred embodiment the invention provides an assay comprising exposing spermatozoa to a tetrazolium dye (preferably MTT), solubilizing the spermatozoa to release their contents into the reaction mixture, and measuring the colour change in the reaction mixture as a result of the reduction of the tetrazolium dye, wherein at least some of the steps are carried out at a pH above pH 4.0, and typically at around pH 6.0.
The results of the colorimetric measurement of the dye reduction can be quantified against a standard of known sperm quality assayed by other means.
The sperm quality assessed is typically viability, but indicators of fertilizing ability and other characteristics of the sperm may be obtained from the colourimetric assessment of the dye reduction.
The sperm is typically avian sperm, but the method is suitable for use on other types of sperm, e.g. mammalian sperm.
Certain embodiments of the invention avoid the need for unstable and toxic reagents such as cyanide and/or the requirement for laboratory facilities like centrifuges. The addition of a reagent such as SDS clears the sperm suspension sufficiently for direct reading of the optical density of the colour produced, thereby enabling a test that can be performed on farm premises without laboratory facilities.